Recovery of 3-hydroxypropionic acid

ABSTRACT

A method for recovering a composition enriched in 3-hydroxypropionic acid from a fermentation broth comprising 3-hydroxypropionic acid and/or salts thereof comprises the steps of: (a) providing the fermentation broth having a pH of from about 2 to about 8 comprising 3-hydroxypropionic acid and/or salts thereof (b) acidifying the fermentation broth; (c) reducing the total sulfate ion and phosphate ion (d) distilling the resulting reduced ion aqueous solution and (e) recovering the product.

FIELD OF THE INVENTION

The present invention relates to recovery of 3-hydroxypropionic acidthereof from a fermentation broth. More specifically, the presentinvention relates to recovery of 3-hydroxypropionic acid from afermentation broth by processing steps.

BACKGROUND OF THE INVENTION

Hydroxycarboxylic acid monomers are useful in many applications, and canbe prepared by a number of routes. One method of manufacture includesthe use of fermentation, which can produce a number of fermentationproducts, depending on the fermenting organism selected and otherfactors. See, for example, U.S. Pat. No. 8,337,663.

SUMMARY OF THE INVENTION

3-Hydroxypropionic acid (“3HP”) in particular is a desired material thatis useful for many industrial applications. It has been discovered thatefficient recovery of 3HP in concentrations and desired purity that issuitable for certain industrial applications is challenging. Inparticular, process steps intended to efficiently recover 3HP can leadto recovery solutions that either do not contain an appropriateconcentration of 3HP for the use in a subsequent process, or can lead tointroduction of undesired impurities. For example, it is challenging torecover 3HP in desired concentrations without dehydrating the 3HP toform acrylic acid in undesirable concentrations. This is particularlythe case when recovering 3HP on a commercially viable scale. Separationsand systems that are suitable for use on the lab bench may not befeasible for use at commercial production levels, and introduction ofdifferent techniques on scale-up introduce new challenges and unexpectedresults. The present invention provides an advantageous process forrecovery of 3HP.

Specifically, a method is provided for recovering a composition enrichedin 3-hydroxypropionic acid from a fermentation broth comprising3-hydroxypropionic acid and/or salts thereof. The method comprises thesteps of:

(a) providing the fermentation broth having a pH of from about 2 toabout 8 comprising:

-   -   3-hydroxypropionic acid and/or salts thereof, and    -   a total sulfate ion and phosphate ion concentration;

(b) acidifying the fermentation broth to lower the pH to from about 1 toabout 3 to form an aqueous solution comprising 3-hydroxypropionic acid;

(c) reducing the total sulfate ion and phosphate ion concentration ofthe aqueous solution to produce a reduced ion aqueous solutioncomprising 3-hydroxypropionic acid;

(d) distilling the reduced ion aqueous solution at a pH of from about 1to about 3 by applying vacuum and heat to the reduced ion aqueoussolution to form an aqueous distillation product comprising3-hydroxypropionic acid; and

(e) recovering the aqueous distillation product comprising3-hydroxypropionic acid at a concentration of at least thirty percent byweight of the aqueous distillation product and wherein the distillationproduct comprises less than five parts by weight acrylic acid per onehundred parts by weight 3-hydroxypropionic acid present.

In some aspects, the fermentation broth comprises a total sulfate ionand phosphate ion concentration of at least 50 ppm, for example, asulfate ion and phosphate ion concentration of at least 100 ppm, atleast 200 ppm, or at least 400 ppm. In some aspects the fermentationbroth comprises a sulfate ion and phosphate ion concentration of atleast 4,000 ppm.

In some aspects the total sulfate ion and phosphate ion concentration ofthe reduced ion aqueous solution from step (c) is less than 15,000 ppm,for example: less than 10,000 ppm, less than 5,000 ppm, less than 3,000ppm, less than 2,500 ppm, less than 2,000 ppm, less than 1,500 ppm totalsulfate ion and phosphate ion concentration, and when particularly lowion levels are desirable, a total sulfate ion and phosphate ionconcentration of less than 1,000 ppm.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate several aspects of the inventionand together with a description of the embodiments serve to explain theprinciples of the invention. A brief description of the drawings is asfollows:

FIG. 1 is a process flow diagram of one embodiment of the presentmethod.

FIG. 2 is a process flow diagram depicting the processing stepsdescribed in Example 11.

FIG. 3 is a process flow diagram depicting the processing stepsdescribed in Example 12.

FIG. 4 is graph showing the effect of adding steam to a distillationsystem that is being fed a reduced ion aqueous solution containing 60percent by weight 3HP.

DETAILED DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS

The embodiments of the present invention described below are notintended to be exhaustive or to limit the invention to the precise formsdisclosed in the following detailed description. Rather a purpose of theembodiments chosen and described is so that the appreciation andunderstanding by others skilled in the art of the principles andpractices of the present invention can be facilitated.

3-Hydroxypropionic acid and/or salts thereof is generated by afermentation process using known fermentation techniques. For purposesof the present discussion, 3-hydroxypropionic acid and/or salts (andsimilar statements such as “3HP (and salts thereof)”, etc) means thatthe compound 3-hydroxypropionic acid is present either in its acid formor in a salt form or in a mixture of the acid form and the salt form.The salt form may comprise one or more counterions.

In step (a) of the present process, a fermentation broth is providedhaving a pH of from about 2 to about 8 comprising 3-hydroxypropionicacid and/or salts thereof, and a total sulfate ion and phosphate ionconcentration of typically at least 100 ppm, for example at least about200 ppm, at least 400 ppm, and in some instances at least about 4,000ppm. In an embodiment, the fermentation broth has a pH of from about 2to about 5 in step (a). In another embodiment, the fermentation brothhas a pH of from about 2.5 to about 4.5 in step (a). Lower pH levels ofthe fermentation broth are advantageous for certain fermentationorganisms that can ferment at commercially acceptable rates at low pH.

In an embodiment, the concentration of 3-hydroxypropionic acid and/orsalts thereof in the fermentation broth of step (a) is from about 30 toabout 200 grams of 3-hydroxypropionic acid equivalents per liter ofbroth. In another embodiment, the concentration of 3-hydroxypropionicacid and/or salts thereof in the fermentation broth of step (a) is fromabout 60 to about 150 grams of 3-hydroxypropionic acid equivalents perliter of broth. In another embodiment, the concentration of3-hydroxypropionic acid and/or salts thereof in the fermentation brothof step (a) is from about 70 to about 130 grams of 3-hydroxypropionicacid equivalents per liter of broth.

In step (b) of the present process, the fermentation broth is acidifiedto lower the pH to from about 1 to about 3 to form an aqueous solutioncomprising 3-hydroxypropionic acid. It has been found that reducing thepH of the aqueous solution comprising 3-hydroxypropionic acid providesprocessing benefits when 3-hydroxypropionic acid is in the acid form,and is beneficial for the recovery of 3-hydroxypropionic acid. In anembodiment, the pH of the fermentation broth is lowered to a pH of fromabout 1.5 to about 2.5 in step (b), and in some aspects from a pH of 1.5to a pH of 2.0.

During the fermentation process, various ingredients are added to thefermentation broth to establish and maintain favorable nutrition and pHconditions to support the particular organism carrying out thefermentation. After completion of the fermentation, various ionicspecies are present that are desirable to remove. The removal of certainions is facilitated in the acidification step through the formation ofinsoluble and/or easily isolatable compounds. For example, undesiredcalcium present in the fermentation broth is removed by lowering the pHthrough the addition of H₂SO₄ or other acidic compounds that will forminsoluble and/or easily isolatable compounds with calcium. Addition ofH₂SO₄ is preferred because the resulting isolatable compound is gypsum.The insoluble and/or easily isolatable compounds are removed byconventional equipment, such as use of a centrifuge, a belt filter, adrum filter, or membrane filter, or other appropriate separationtechniques.

In an embodiment of the present invention, cells from the fermentationprocess are preferably removed from the fermentation broth prior toaddition of acid, so the amount of biological materials present in thegypsum or like material that is removed is below the amount suitable forend use of this product. Alternatively, the cells may be removed afterthe acidification step (b), together with the removal of insolubleand/or easily isolatable compounds that may be formed during theacidification step.

In step (c) of the present process, the total sulfate ion and phosphateion concentration of the aqueous solution is reduced. Typically, thetotal sulfate ion and phosphate ion concentration of the reduced ionaqueous solution produced in step (c) is less than 15,000 ppm. Reductionof the ion content in the aqueous solution prior to distillation isadvantageous, because it has been found that carrying out these steps inthis order reduces the amount of undesired side products and impuritiespresent in the final recovered distillation product. In particular,reduction of the ion content of the aqueous solution prior todistillation beneficially reduces the amount of acrylic acid formed insubsequent steps of the 3-hydroxypropionic acid recovery process, andalso reduces the boiling point of the reduced ion aqueous solution, andreduces the energy needed to recovery the 3HP. In an embodiment, thetotal sulfate ion and phosphate ion concentration is reduced to lessthan about 10,000 ppm, less than about 5000 ppm, less than about 3000ppm, less than 2,500 ppm, less than 2,000 ppm, less than 1,500 ppm, andwhen particularly low ion levels are desirable, a total sulfate ion andphosphate ion concentration of less than 1,000 ppm in step (c). In anembodiment, the step (c) of reducing the total sulfate ion and phosphateion concentration of the aqueous solution is carried out by utilizing anion exchanger. In an embodiment, the ion exchanger comprises an anionexchange resin. In an embodiment, the amount of positively charged ionsin the aqueous solution is reduced by use of an ion exchanger thatcomprises a cation exchange resin. In an embodiment of the presentinvention, negative ion reduction has a tendency to increase the pH ofthe aqueous solution, which is preferably at least partiallycounteracted by reduction in pH by reducing the amount of positive ionsin the aqueous solution. In an embodiment of the present invention, thepH of the aqueous solution that is distilled in step (d) is adjustedduring the ion reducing step (c).

In step (d) of the present process, the reduced ion aqueous solution isdistilled by applying vacuum and heat to the reduced ion aqueoussolution to form an aqueous distillation product comprising3-hydroxypropionic acid. The use of “more gentle” distillationtechniques involving application of vacuum and lower levels of heat thanwould otherwise be required in distillation process undertaken atstandard pressure is advantageous because it reduces the likelihood offormation of undesired side reactants in the recovery process. Inembodiments, the distillation step (d) comprises heating the reduced ionaqueous solution to a temperature of from about 100° C. to about 200°C.; or from about 120° C. to about 180° C., or from about 130° C. toabout 170° C., or from about 130° C. to about 150° C. In embodiments,the distillation step (d) is carried at a pressure of from about 0.5 toabout 50 mm Hg absolute, or at a pressure of from about 5 to about 40 mmHg absolute, at a pressure of from about 10 to about 35 mm Hg absolute.

Distillation by exposing the reduced ion aqueous solution to elevatedtemperature for relatively short times is also advantageous for reducingthe likelihood of formation of undesired side reactants in the recoveryprocess. In embodiments, the distillation step (d) is carried out byheating the reduced ion aqueous solution at a temperature above 100° C.for a time less than five minutes, or at a temperature above 120° C. fora time less than three minutes, or at a temperature above 130° C. for atime less than two minutes.

In embodiments, the distillation step (d) may be carried out by shortresidence time distillation techniques. In embodiments, the distillationstep (d) is carried out by equipment selected from wiped filmevaporation equipment, rising film evaporator equipment, thin filmevaporation equipment, (centrifugal) molecular distillation equipment,falling film distillation equipment, or combinations, thereof. See ageneral discussion of such equipment in U.S. Pat. No. 7,560,579.

The concentration of 3-hydroxypropionic acid in the reduced ion aqueoussolution prior to distillation of step (d) may have an impact on thepresence of one or more undesirable side products or other impurities inthe final concentrated product. In an embodiment, the concentration of3-hydroxypropionic acid in the reduced ion aqueous solution prior todistillation of step (d) is from about 20 percent by weight to about 80percent by weight. In another embodiment, the concentration of3-hydroxypropionic acid in the reduced ion aqueous solution prior todistillation of step (d) is from about 25 percent by weight to about 70percent by weight. In another embodiment, the concentration of3-hydroxypropionic acid in the reduced ion aqueous solution prior todistillation of step (d) is from about 30 percent by weight to about 60percent by weight.

In an embodiment of the present invention, the distillation step (d) ispreferably carried out at a pressure of from about 20 to about 35 mm Hgabsolute and a temperature of from about 130° C. to about 150° C. at a3-hydroxypropionic acid concentration of from about 30 percent by weightto about 60 percent by weight.

In step (e) of the present process, the aqueous distillation product isrecovered that comprises 3-hydroxypropionic acid typically at aconcentration of at least thirty percent by weight of the aqueousdistillation product, wherein the aqueous distillation product typicallycomprises less than five parts by weight acrylic acid per one hundredparts by weight 3-hydroxypropionic acid present. In embodiments, the3-hydroxypropionic acid is recovered in step (e) at a concentration ofat least about thirty-five percent by weight of the aqueous distillationproduct, or at a concentration of at least about forty percent by weightof the aqueous distillation product. In embodiments, the aqueousdistillation product recovered in step (e) contains less than aboutthree parts by weight acrylic acid per one hundred parts by weight3-hydroxypropionic acid present, or less than about 1 part by weightacrylic acid per one hundred parts by weight 3-hydroxypropionic acid, orless than about 0.5 parts by weight acrylic acid per one hundred partsby weight 3-hydroxypropionic acid.

In embodiments of the present invention, the concentration of3-hydroxypropionic acid in the aqueous distillation product comprising3-hydroxypropionic acid recovered in step (e) is from about 30 percentby weight to about 80 percent by weight; or is from about 30 percent byweight to about 70 percent by weight; or is from about 30 percent byweight to about 60 percent by weight.

It will be noted that over time in storage, 3-hydroxypropionic acid maytend to react, for example to form dimers and trimers. The concentrationof 3-hydroxypropionic acid that is recovered in step (e) is determinedimmediately after completion of the recovery process, prior to storagethat may result in reactions of 3-hydroxypropionic acid that wouldchange the determination of recovery concentration.

Combinations of the specific embodiments of each of the steps (a)-(e) asidentified above are additionally contemplated. For example (and withoutlimiting the contemplated combination of the various specificembodiments), in a particularly advantageous embodiment of the presentinvention, the method comprises the steps wherein the total sulfate ionand phosphate ion concentration is reduced to less than about 5000 ppmin step (c); the concentration of 3-hydroxypropionic acid in the aqueousdistillation product of step (d) is from about 30 percent by weight toabout 60 percent by weight; and the aqueous distillation productrecovered in step (e) contains less than about 1 part by weight acrylicacid per one hundred parts by weight 3-hydroxypropionic acid. In anotherparticularly advantageous embodiment of the present invention, themethod comprises the steps wherein the total sulfate ion and phosphateion concentration is reduced to less than about 3000 ppm in step (c);the concentration of 3-hydroxypropionic acid in the aqueous distillationproduct of step (d) is from about 30 percent by weight to about 60percent by weight; and the aqueous distillation product recovered instep (c) contains less than about 0.5 part by weight acrylic acid perone hundred parts by weight 3-hydroxypropionic acid.

In another particularly advantageous embodiment of the presentinvention, the method comprises the steps wherein:

the total sulfate ion and phosphate ion concentration is reduced to lessthan about 3000 ppm in step (c);

the concentration of 3-hydroxypropionic acid in the reduced ion aqueoussolution prior to distillation of step (d) is from about 30 percent byweight to about 60 percent by weight;

the distillation step (d) comprises heating the reduced ion aqueoussolution to a temperature of from about 130° C. to about 150° C.;

the distillation step (d) is carried out at a pressure of from about 10to about 35 mm Hg absolute; and

the aqueous distillation product recovered in step (e) contains lessthan about 0.5 parts by weight acrylic acid per one hundred parts byweight 3-hydroxypropionic acid.

In an embodiment of the present invention, the method further comprisesnew steps (f) of recovering a distillation bottom stream from distillingstep (d), the distillation bottom stream comprising from about 5 percentto about 20 percent 3-hydroxypropionic acid of the total amount of the3-hydroxypropionic acid present in the reduced ion aqueous solution ofstep (c);

(g) applying vacuum and heat to the distillation bottom stream; and

(h) recovering a second aqueous distillation product comprising3-hydroxypropionic acid, wherein the second aqueous distillation productcomprises less than about 3 part by weight acrylic acid per one hundredpart by weight 3-hydroxypropionic acid present in the second aqueousdistillation product.

This embodiment provides enhanced recovery of 3-hydroxypropionic acidfrom the aqueous distillation product by not requiring aggressivedistillation conditions to be applied to the entire reduced ion aqueoussolution. Additionally, more total 3-hydroxypropionic acid is recoveredfrom the reduced ion aqueous solution to be distilled as compared to alike process where only one distillation step is carried out. In anembodiment, water is added to the distillation bottom stream prior tostep (g). This embodiment enhances overall recovery of3-hydroxypropionic acid by making it easier to recover3-hydroxypropionic acid from the second distillation step.

It should be noted that a great portion of the water present in thereduced ion aqueous solution that is distilled in step (d) is removedwith the 3-hydroxypropionic acid that is recovered in step (e), andtherefore the distillation bottom stream initially has a relatively high3-hydroxypropionic acid. Dilution of the distillation bottom stream withwater helps reduce formation of undesired side reactants or impuritiesduring the second distillation, and additionally assists in efficientperformance of the second distillation operation.

In another embodiment of the present invention, the method furthercomprises prior to the acidifying step (b) an additional step (i) ofincreasing the concentration of 3-hydroxypropionic acid and/or saltsthereof in the fermentation broth of step (a) to from about 100 to about500 grams of 3-hydroxypropionic acid equivalents per liter of broth byevaporating a portion of the liquid present in the fermentation broth ata broth temperature of from about 60° C. to about 100° C. In anembodiment, the concentration of 3-hydroxypropionic acid and/or saltsthereof in the fermentation broth is increased to from about 250 toabout 400 grams, and in some aspects from 140 to 400 grams, from 150 to350 grams, from 200 to 330 grams, from 220 to 320 grams, or from 230 to300 grams of 3-hydroxypropionic acid equivalents per liter of broth.

This low heat increase of concentration of 3-hydroxypropionic acid isparticularly advantageous in providing both an economic process and alsoin reducing introduction of undesired side products or impurities. In anembodiment, the evaporation of liquid takes place at a broth temperatureof from about 70° C. to about 90° C. In an embodiment, the evaporationof liquid takes place at a pressure of from about 250 to about 300 Torr.

In another embodiment of the present invention, the method furthercomprises prior to the distilling step (d) the new step (j) ofincreasing the concentration of 3-hydroxypropionic acid in the reducedion aqueous solution of step (c) to from about 30 percent by weight toabout 70 percent by weight of the solution by evaporating a portion ofthe liquid present in the reduced ion aqueous solution at a solutiontemperature of from about 60° C. to about 100° C. As above, this lowheat increase of concentration of 3-hydroxypropionic acid isparticularly advantageous in providing both an economic process and alsoin reducing introduction of undesired side products or impurities. In anembodiment, the evaporation of liquid takes place at a solutiontemperature of from about 70° C. to about 90° C. In an embodiment, theevaporation of liquid takes place at a pressure of from about 250 toabout 300 Torr.

In an embodiment, the present method comprises both steps (i) and (j)discussed above.

In another embodiment of the present invention, the method furthercomprises the new step (k) of reducing the amount of micro-organismcells present in the fermentation broth prior to step (c). In anembodiment, the reducing step (k) takes place prior to the acidifyingstep (b). In an embodiment, the reducing step (k) is carried out by useof a separator device selected from a centrifuge, a belt filter, a drumfilter, and a membrane filter.

FIG. 1 is a process flow diagram of one embodiment of the presentmethod. As shown, a fermentation broth is provided, and cells areremoved therefrom. Water is removed by a first evaporation step and thefermentation broth is acidified to form an aqueous solution comprising3-hydroxypropionic acid. Gypsum is removed and the total sulfate ion andphosphate ion concentration of the aqueous solution is reduced using ionexchangers. The reduced ion aqueous solution comprising3-hydroxypropionic acid is subjected to a second evaporation step toremove water, and the reduced ion aqueous solution is subjected to twodistillation steps.

In the first evaporation step described above, the concentration of3-hydroxypropionic acid and/or salts thereof are after the completion ofthe first evaporation step typically less than 350 grams3-hydroxypropionic acid equivalents per liter.

EXAMPLES

Representative embodiments of the present invention will now bedescribed with reference to the following examples that illustrate theprinciples and practice of the present invention.

The following analytical methods and sample preparations were used inthe examples below.

Analytical Method for 3HP

3-Hydroxypropionic acid (3HP), acrylic acid, and various other organicacids, alcohols, and sugars in the samples are analyzed using highperformance liquid chromatography (HPLC). This HPLC method utilizes acombination of two BioRad Aminex HPX-87H columns, in conjunction withRefractive Index (RI) detection and Ultraviolet (UV) detection at 210nm. The RI detector is for the quantification alcohols and sugars, andthe UV detector for all the organic acids. Standards and samples areprepared by mass in volumetric flasks diluted with the mobile phase. Nointernal standard is used. Results were calculated in weight %.

The HPLC is a Waters Alliance 2695 modular High Performance LiquidChromatography system that includes a pump, auto-sampler, solventin-line gasser, and column heater. The RI detector is a Waters 2410Refractive Index Detector, and the UV detector a Waters 2487 DualWavelength Ultraviolet Detector. The columns are Aminex HPX-87H 300×7.8mm columns (BioRad), used with a Security Guard cartridge holder(Phenomenex) and Carbo H+ guard cartridges (Phenomenex).

An Isocratic mobile phase of 10 mM H₂SO₄ in high-purity water,containing sodium azide (0.005%), filtered through an 0.45 micronfilter, is used at a flow rate of 0.5 mL/min. The column temperature is55° C. Sample injection volume is 20 μL. Each run is 60 minutes long.The internal temperature of the Refractive index detector is 35° C. 18+megaohm ultrapure water is used.

Standard Stock Solution 1 containing Glucose (0.1 g/L), Malic acid (0.1g/L), Pyruvic acid (0.1 g/L), Arabitol (0.1 g/L), Succinic acid (0.1g/L), Lactic acid (0.1 g/L), Glycerol (0.1 g/L), and Acrylic acid (0.1g/L) in 10 mM H2SO4 is prepared. The stock solution is stored in arefrigerator, and diluted 10:1 with 10 mM H2SO4 solution to prepare theStandard 1 working solution for HPLC analysis. Standard 3HP (5 g/L) isprepared in 10 mM H2SO4 solution. This is the 3HP working standardsolution for HPLC analysis.

For sample preparation, 0.25 grams of sample is weighed into a 25-mlvolumetric flask and diluted with 10 mM sulfuric acid and filteredthrough a 0.45 micron nylon syringe filter. The wt. % of alcoholsincluding glycerol and sugars are calculated using Refractive Index(RID) peak areas. The wt. % of organic acids is calculated using UV peakarea.

The phosphate and sulfate ions are determined by measuring elementalsulfur and phosphorous by inductively coupled plasma atomic emissionspectroscopy (ICP) analysis. Analysis was done using a Spectro ArcosFHS12 instrument. All sulfur and phosphorous is believed to be in theform of sulfate and phosphate ions, respectively.

General Procedures for Examples 1 Through 8 3HP Fermentation Broth

3HP broth produced by fermentation from glucose in yeast is used as thestarting material for the processing steps. The fermentation brothcontains 37 g/L 3HP in addition to other fermentation by-productsincluding unfermented sugars, other organic acids such as lactic,pyruvic, succinic, and salts. Some of the major components are shown inTable 1 below. Yeast biomass is also removed from the broth using benchtop centrifugation (2200×g for 5 minutes) and then the supernatant isdecanted.

TABLE 1 Typical concentrations of major components of the aqueous 3HPfermentation broth. Component Concentration (g/L) 3HP 35 Unfermentedsugars 1 Organic acids 3 Calcium 6 Phosphate 4 Glycerol <10 Typical pH2-5

Acidulation

The calcium in the broth is removed by adding concentrated H₂SO₄ untilthe pH of the broth solution is between 2-2.5. The precipitated gypsumis removed using bench top centrifugation (2200×g for 5 minutes) and thesupernatant is then decanted.

Ion Exchange (Demineralization)

The acidulated fermentation broth goes through a demineralization stepusing cation and anion exchange. Cation exchange is done using AMBERLITEIR120 Strong Acid Cation Exchanger resin, available from Rohm and Haas.The resin is loaded in a 1″ internal diameter column with an approximatebed volume of 200 ml. The column is conditioned by passing 200 ml of 1MHCl through the column followed by DI water until the effluent pH isabove 5. The cation column is used to reduce the amounts of calcium,sodium, potassium, iron and magnesium. 3HP broth is passed through thecolumn until breakthrough conditions (cation resin capacity is exceeded)are reached indicated by a rise in pH in the effluent stream. Dependingon the ionic loading, 1-10 L of broth is passed through the column.

The effluent from the cation exchange is then passed through anionexchange column. The anion exchange resin is DOWEX MARATHON WBA WeakBase Anion Exchange resin available from The Dow Chemical Company. Theresin is loaded in a 1″ internal diameter column with an approximate bedvolume of 200 ml. The column is conditioned by passing 200 ml of 1M NaOHthrough the column followed by DI water until the effluent pH is below8. The anion column is used to reduce the amounts of sulfate, phosphateand chloride ions. 3HP cation exchange effluent is passed through thecolumn until breakthrough conditions (anion resin capacity is exceeded)are reached indicated by a drop in pH in the effluent stream. Dependingon the ionic loading, 0.5-2 L of broth are passed through the column.

Evaporation/Concentration

The material is concentrated using a rotary evaporator to a 3HPconcentration of 550-650 g/L. The pressure is set at 50 torr and thetemperature increases from 40 to 60° C. as the material becomes moreconcentrated. The concentration step can take place before and/or afterthe demineralization steps.

Distillation

Distillation was carried out using a short path wiped film evaporator(model KDL4 manufactured by UIC). Material is fed from the top of theunit down to the heated surface. The evaporator, with a total surfacearea of 0.043 m², is a jacketed borosilicate glass cylinder and uses hotoil to control temperature. Inside the heated surface, teflon rollersare used to distribute the feed material. For feed rates of 4-7 ml/min,the residence time or contact time of the feed material to the heatedsurface is approximately 30-60 seconds. An internal condenser located inthe center of the heated surface is used to condense the distillatephase which is collected in a receiver flask. The non-volatile bottomsare collected in a separate receiver flask. The feed rate is controlledby a peristaltic pump. Feed rates are measured by measuring the weightsof the feed flask over time. Typical feed weights are around 50-100 g.In the following examples, the feed rate, jacket temperature, and vacuumpressure are controlled. All pressures are absolute. Unless otherwiseindicated, reported distillation temperatures are jacket temperatures.

Example 1 (Comparative)

This example shows the effect of having a high combined sulfate andphosphate concentrations (for example, above 100,000 ppm) in the reducedion aqueous solution of 3HP feeding the distillation results in arecovered distillate with a high content of acrylic acid (for example,greater than 30 parts Acrylic Acid per 100 parts 3HP).

3HP broth is prepared as described above in the General Procedure. Thefermentation broth has a pH of 4 and contains 37 g/L 3HP, 5927 ppmcalcium, 243 ppm sulfate, and 8946 ppm phosphate. The fermentation brothundergoes cell separation by centrifugation. The broth is then acidifiedwith concentrated sulfuric acid to pH of 2 and then clarified usingcentrifugation to remove gypsum and other un-dissolved solids. Thecalcium concentration of the acidified and clarified broth is 524 ppm.The broth is demineralized using cation and anion exchange columns. Thesulfate concentration is reduced to 1583 ppm and the phosphateconcentration is reduced to 5998 ppm with a 3HP concentration close to35 g/L. The resulting 3HP solution is then concentrated by evaporationto 638 g/L which also increased the sulfate concentration to 33466 ppmand phosphate concentration to 95473 ppm.

The reduced ion aqueous solution of 3HP is then distilled using thewiped film evaporator (WFE). The distillation conditions are shown inTable 1-1 which also summarizes the relation between the distillationfeed, conditions and acrylic acid formation.

TABLE 1-1 Distillation conditions using Wiped Film Evaporator. Recoveryis calculated as the amount of 3HP collected in the distillate streamcompared to the 3HP fed into the WFE. Both 3HP and Acrylic Acidconcentrations are given along with the parts Acrylic Acid per 100 parts3HP. SO₄ ²⁻ and PO₄ ³⁻ concentrations are calculated from sulfur andphosphorous measured by ICP, respectively. Distillate ProfileDistillation Parameters Feed Concentration Parts Acrylic Temp Pres Feed3HP SO4 PO4 Recovery [3HP] [AA] Acid per 100 ID (° C.) (torr) (g/min)(g/L) (ppm) (ppm) (%) (g/L) (g/L) Parts 3HP Ex1 180 20 6 638 33466 9547312% 235 91 39.1

Example 2

This example shows the effect of lowering the combined sulfate andphosphate concentrations (for example, below 15,000 ppm) in the reducedion aqueous solution of 3HP fed into distillation results in a recovereddistillate with lower acrylic acid formation (for example, below 3 partsper 100 parts 3HP).

3HP broth is prepared as described above in the General Procedure. Thefermentation broth has a pH of 4 and contains 37 g/L 3HP, 4409 ppmcalcium, 224 ppm sulfate, and 7654 ppm phosphate. The fermentation brothundergoes cell separation by centrifugation. The broth is then acidifiedwith concentrated sulfuric acid to pH of 2 and then clarified usingcentrifugation. The calcium concentration of the acidified and clarifiedbroth is 427 ppm. The broth is demineralized using cation and anionexchange columns. The resulting 3HP solution is then concentrated byevaporation to approximately 600 g/L 3HP which also results in sulfateconcentration of 30025 ppm and phosphate concentration of 22241 ppm. Theconcentrated solution is demineralized a second time by passing throughcation and anion exchange columns again. The resulting concentratedsolution has a sulfate concentration of 1972 ppm and phosphateconcentration of 11477 ppm with a 3HP concentration of 556 g/L.

The prepared broth is then distilled using the wiped film evaporator(WFE). The distillation conditions are shown in Table 2-1 which alsosummarizes the relationship between the distillation feed, conditionsand acrylic acid formation.

TABLE 2-1 Distillation conditions using Wiped Film Evaporator. Recoveryis calculated as the amount of 3HP collected in the distillate streamcompared to the 3HP fed into the WFE. Both 3HP and Acrylic Acidconcentrations are given along with the parts Acrylic Acid per 100 parts3HP. SO₄ ²⁻ and PO₄ ³⁻ concentrations are calculated from sulfur andphosphorous measured by ICP, respectively. Distillate profileDistillation Parameters Feed Concentration Parts Acrylic Temp Pres Feed3HP SO4 PO4 Recovery [3HP] [AA] Acid per 100 ID (° C.) (torr) (g/min)(g/L) (ppm) (ppm) (%) (g/L) (g/L) Parts 3HP Ex2 180 20 6.5 556 197211477 75% 673 8.5 1.31

Example 3

This example shows the recovery of 3HP on a larger scale going throughthe process of cell removal, acidulation, ion exchange and distillation.The reduced ion aqueous solution of 3HP has a lower combined sulfate andphosphate concentration (for example, below 2000 ppm).

3HP is prepared as described in the General Procedure. The fermentationbroth undergoes cell separation by membrane filtration using a ceramicmembrane with a 0.1 micron pore size. The broth is acidified withconcentrated sulfuric acid to pH of 2.6 and is then clarified using afilter. The broth is demineralized using cation and anion exchangecolumns and then concentrated up to 37.2% monomer 3HP by evaporation.The reduced ion aqueous solution having a phosphate and sulfateconcentration of 1088 ppm and 459 ppm, respectively, feeds into a risingfilm evaporator (RFE) for distillation.

The RFE is a single tube (1.5″×20′) with a steam jacket. The feed ispumped to the bottom of the tube where the 3HP, water and other volatilecomponents vaporize and carry over to an external condenser where thedistillate stream condenses and is collected. The material that does notvolatilize flows over the top of the RFE and is collected separately asa bottoms residue. For this example, the feed rate is set at 75 g/minresulting in a residence time in the heated tube of approximately 90seconds. The vacuum pressure is set at 27 torr and the steam pressure inthe jacket is set to 75 psig, resulting in an RFE wall temperature of160° C.

TABLE 3-1 Distillation conditions using Rising Film Evaporator. Recoveryis calculated as the amount of 3HP collected in the distillate streamcompared to the 3HP fed into the RFE. Both 3HP and Acrylic Acidconcentrations are given along with the parts Acrylic Acid per 100 parts3HP. SO₄ ²⁻ and PO₄ ³⁻ concentrations are calculated from sulfur andphosphorous measured by ICP, respectively. Distillate profileDistillation Parameters Feed Concentration Parts Acrylic Temp Pres Feed3HP SO4 PO4 Recovery [3HP] [AA] Acid per 100 ID (° C.) (torr) (g/min)(wt %) (ppm) (ppm) (%) (wt %) (wt %) Parts 3HP Ex3 160 27 75 37.2 4591088 77% 66 0.03 0.04

This data shows 3HP can be recovered with a very low Acrylic Acidcontent (below 1 part in 100 3HP, and even below 0.5 parts in 100 parts3HP) when the combined sulfate and phosphate concentration in thereduced ion aqueous solution feeding the distillation is low.

Example 4

This example shows additional 3HP can be recovered by distilling thebottoms residue from the first distillation in Example 3.

The bottoms residue collected from Example 3 is distilled using a wipedfilm evaporator as described in the General Procedure. This bottomsstream is 5-6 times more concentrated in sulfates and phosphatescompared to the reduced ion aqueous 3HP solution that feeds the RFE inExample 3, as the majority of the more volatile water and 3HP iscollected in the distillate stream. The material is fed into the WFE ata rate of 5 g/min at a pressure of 1 torr and jacket temperature of 180°C. The second distillate stream collected contains 507 g/L 3HP and 12.9g/L acrylic acid (2.5 parts acrylic acid per 100 parts of 3HP). Thetotal 3HP recovery increases from 77% (single RFE distillation) to 84%(combined RFE and WFE) based on the reduced ion aqueous solution.

Example 5

This example demonstrates the effects of distillation time, temperature,3HP concentration, and anion concentration on the length of time ofdistillation that can be used without creating high quantities ofacrylic acid.

In this example, 3HP broth is prepared through theEvaporation/Concentration step as described in the General Procedure.Following the Ion Exchange step of the General Procedure, the 3HPconcentration in the reduced ion aqueous solution is 30% by weight. TheIon Exchange step is performed at two different conditions. In one casethe total sulfate and phosphate concentration is reduced to 3,000 ppm,and in the second case is reduced to 10,000 ppm. The material is thenconcentrated to different levels as described in theEvaporation/Concentration step of the General Procedure to threedifferent levels of 3HP concentration.

Table 5-1 shows typical parameters of concentration of 3HP, the combinedsulfate and phosphate level in the reduced ion aqueous solution feedingthe distillation, and the time at elevated temperatures in distillationthat are believed will result in an acrylic acid formation of less than1 part per 100 parts 3HP in the distillate.

TABLE 5-1 Distillation Temperature 3HP Concentration Allowed Time(Degrees C.) (g/L) SO4 + PO4 (ppm) (min) 140 500 5,000 3 150 600 6,0001.5 130 300 10,000 4

By performing distillation at lower 3HP concentrations, the rate ofacrylic acid is reduced. Additionally, the boiling point of the reducedion aqueous solution is lower at lower 3HP and concentrations, so thatthe temperature at which distillation can be carried out for comparablerecoveries is lower. These lower temperatures further reduce the rate ofacrylic acid formation.

Example 6

This example shows the effect of temperature in distillation on theacrylic acid formed.

3HP broth is prepared as described above in the General Procedure. Thefermentation broth has a pH of 4 and 37 g/L 3HP. The fermentation brothundergoes cell separation by centrifugation. The broth is evaporated toreach a concentration of 220 g/L 3HP resulting in a sulfateconcentration of 2383 ppm and phosphate concentration of 7276 ppm. Noacidulation is done in this example. A similar result to acidulating thebroth is achieved by increasing the capacity of the cation exchangeresin to remove the calcium ions that would be removed by acidulation.The broth is then demineralized through ion exchange which reduces thesulfate concentration to 461 ppm and phosphate concentration of 124 ppm.The 3HP solution is then concentrated to 577 g/L 3HP, 1755 ppm sulfate,and 472 ppm phosphate.

The prepared broth is then distilled using the wiped film evaporator(WFE). The distillation conditions are shown in Table 6-1, which alsosummarizes the relationship between the distillation feed, distillationconditions and acrylic acid formation.

TABLE 6-1 Distillation conditions using Wiped Film Evaporator. Recoveryis calculated as the amount of 3HP collected in the distillate streamcompared to the 3HP fed into the WFE. Both 3HP and Acrylic Acidconcentrations are given along with the parts Acrylic Acid per 100 parts3HP. SO₄ ²⁻ and PO₄ ³⁻ concentrations are calculated from sulfurmeasured by ICP, respectively, Distillate profile DistillationParameters Feed Concentration Parts Acrylic Temp Pres Feed 3HP SO4 PO4Recovery [3HP] [AA] Acid per 100 ID (° C.) (torr) (g/min) (g/L) (ppm)(ppm) (wt %) (g/L) (g/L) Parts 3HP Ex6a 160 20 5 577 1755 472 85% 6691.4 0.21 Ex6b 170 20 5 577 1755 472 78% 681 3.2 0.45 Ex6c 180 20 5 5771755 472 68% 608 5.2 0.84

The results show temperatures below 180° C. (e.g. 160° C. and 170° C.)results in Acrylic Acid formation less than 0.5 parts per 100 parts 3HP(i.e. wt % acrylic acid less than 0.5 wt %)

Example 7

This example is similar to Example 4 and shows the amount of acrylicacid formed in the second distillate stream is lowered as compared toExample 4 by adding additional water to the feed of the seconddistillation.

Water is added to the Bottoms residue generated in Example 3 to increasethe water concentration to 20 wt %. This diluted stream feeds into a WFEdistillation at 5 g/min. The WFE operates at 160° C. and 20 torr. Theresulting second distillate has a 1.1 parts Acrylic Acid per 100 parts3HP.

Example 8 (Comparative)

This example shows how large amounts of acrylic acid is generated withlong exposure time to heat.

The same 3HP broth prepared and used in Example 6 is used in thisexample, except for the final WFE distillation. Instead, a batchdistillation experiment is set up using a 500 ml round bottom flask tohold the material. The flask is placed in an electric heating mantle toprovide heat. Above the flask is an adapter to connect the condenser(water cooled) which allows the vapor to cool and collect in a separatedistillate collection flask. A thermometer is placed at the adaptor tomeasure the temperature of the vapor just before entering the condenser.The setup is connected to a vacuum pump set at 20 torr.

200 g of the prepared broth is added to the flask and heating mantle isturned on. Heating takes approximately 2 hours to reach a liquidtemperature of approximately 150° C. At that point condensate startscollecting in the distillate collection flask and the vapor temperaturereaches 140° C. Distillation continues for 30 minutes at which time thevacuum is shut off and the heating mantle is removed. 83.6 g ofdistillate is collected and 116.5 g of bottoms is collected. Table 8-1shows the composition of the 3 streams.

TABLE 8-1 Parts Acrylic Acrylic Acid to 100 Sample 3HP (g/L) (g/L)3HPRecovery Parts 3HP Feed 577 ND* Distillate 184 12 13% 6.6 Bottoms 88711 1.1 *ND indicates not detectable by HPLC.

Example 9

Fermentation:

Referring to FIG. 1, this example shows a large scale fermentationprocess for producing fermentation broth containing 3HP and/or saltsthereof.

A 3HP producing yeast strain is employed in a fermentation to produce3HP and/or salts thereof. Fermenters are inoculated with biomass grownin defined medium see Tables 9-1, 9-2 and 9-3. 3HP producing yeaststrains are multiplied (i.e. grown) in a 10,000 liter (L) aerobic seedfermentor. The resulting yeast producing strains are inoculated into a1,000,000 liter production fermenter to provide an initial cell dryweight of 0.1 g/L. The fermenter media at the time of inoculation isoutlined in Table 9-1. During the fermentation, pH is controlled at 4.0by addition of an aqueous suspension of 30% Ca(OH)₂ (lime). Theproduction fermenter is sparged at 0.1 VVM (standard volume of airsparged per volume of the fermentor per minute) with air. Agitation isprovided to the production fermenter to achieve an oxygen uptake rate(OUR) of 14 mmol/(L*h). The fermentation is operated so that after thecells achieve a sufficient density, oxygen limitation is achieved andsubsequently maintained throughout the rest of the fermentation (e.g.,dissolved oxygen less than about 10% of air saturation at 1 atm).Dissolved oxygen during the fermentation is measured using MettlerToledo INPRO® 6800 sensor (Mettler-Toledo GmbH, Urdorf, Switzerland).

TABLE 9-1 Defined Media for production fermenters Compound Concentration(g/kg) C₆H₁₂O₆ 107 g/l initial glucose Urea 1.7 KH₂PO₄ 1.5 MgSO₄—7H₂O0.25 1000x Vitamin Solution 1 1000x Trace Solution 1

TABLE 9-2 1000X Trace Solution. Chemical g/1.0 L C₁₀H₁₄N₂Na₂O₈•2H₂O15.00 ZnSO₄•7H₂O 4.50 MnCl₂•4H₂O 1.00 CuSO₄•5H₂O 0.30 FeSO₄•7H₂O 3.00

TABLE 9-3 1000X Vitamin Solution Chemical g/1.0 L Biotin (D−) 0.05 CaD(+) panthotenate 1.0 Nicotinic acid 5.0 Myo-inositol (for 25.0microbiology) Thiamine hydrochloride 1.0 Pyridoxine 1.0 hydrochloridep-Aminobenzoic acid 0.20

Cell concentration at the time of inoculation and at the end offermentation is obtained from an optical density measurement using anestablished conversion factor between dry cell mass and optical density.Optical density is measured at a wavelength of 600 nm with a 1 cmpathlength using a model Genesys20 spectrophotometer (ThermoScientific). Unless explicitly noted otherwise, an experimentallyderived conversion factor of 2.3 OD600 units per 1 g dry cell mass isused to estimate cell dry weight.

OUR is calculated using methods known to those of skill in the art. Forthis example, Oxygen, Nitrogen and CO₂ values are measured by a MassSpectrometer (Thermo Scientific).

The batch finishes and samples are taken at 40 h batch time and analyzedfor biomass growth via OD600. Glucose and 3HP are measured by highperformance liquid chromatography with refractive index detector. Thefinal 3-hydroxypropionic acid equivalents (based on 3HP and/or saltsthereof contained in the fermentation broth) concentration is 80 g/L,which equates to a volumetric productivity of 3HP of 2 g/(L*hr), and ayield of 0.75 g 3-hydroxypropionic acid equivalents per gram glucose anda cell dry weight of 7-10 g/L. Final sulfate and phosphateconcentrations in the broth is 300 ppm and 200 ppm, respectively. Insome instances, with an appropriate organism and fermentationparameters, the final 3HP equivalents concentration may be at least 100g/liter (L), a volumetric productivity of 3HP of 2.5 g/(L*hr), and ayield of at least 0.75 g 3-hydroxypropionic acid equivalents per gram ofglucose (in some instances at least 0.77 g 3-hydroxypropionic acidequivalents per gram of glucose), with a final cell dry weight of lessthan 10 g/L (preferably less than 5 g/L).

Example 10

This example shows the processing of 3HP and/or salts thereof producedfrom the fermentation of Example 9 using cell removal, evaporation,acidulation, clarification, and ion exchange.

Cell Removal:

Referring to step 2 of FIG. 1, the fermentation broth from Example 9 isobtained and is fed into several centrifuges at a combined rate of 283m³/hr. The centrifuges each have a disc stack configuration where thecell free fermentation broth containing 3HP and/or salts thereof passthrough to the product discharge. The cell concentrate underflow streamcontains 40-50 volume percent cells based on the liquid present anddischarges through ejection nozzles on each centrifuge. Concentratingthe cells below 40% by volume of the discharge results in lower recoveryof 3HP from the overall recovery process as a higher percentage of 3HP(and/or salts thereof) ends up in the cell concentrate discharge stream.Concentrating the cells above 50% typically causes the cell concentratestream to be very viscous and difficult to handle. To improve therecovery of 3HP from the overall recovery process, additional water (2-3times the volume of the cell concentrate stream) is mixed with theconcentrate stream and the diluted volumetrically adjusted cellconcentrate stream is fed into the inlet of another centrifuge.Additional cell free 3HP containing broth is discharged; and the cellsare once again concentrated up to 40-50% vol % and discharges throughthe ejection nozzles on the centrifuge. To further improve 3HP recovery,the cell concentrate discharge from the 2^(nd) stage centrifuge isdiluted with water (2-3 times by volume) and is processed through a3^(rd) stage centrifuge. The three cell free 3HP containing brothstreams are combined and then carried forward for further processing.

In order to reduce the amount of wash water used, a counter current washsystem is employed. Fresh water combines with the cell concentratestream exiting the 2^(nd) stage centrifuge and feeding the 3^(rd) stagecentrifuge. The cell free discharge of the 3^(rd) stage centrifuge isused to dilute the cell concentrate discharge of the 1^(st) stagecentrifuge and then feeds the 2^(nd) stage centrifuge. After cellseparation, the concentration of 3HP (and/or salts thereof) drops tobetween 90-95% of the starting concentration of the 3HP and/or saltthereof in the fermentation broth from Example 9, depending on how muchwash water is used.

Evaporation:

Referring to FIG. 1, step 3, the cell free 3HP containing combined brothis evaporated to raise the concentration of 3HP (and/or salts thereof).The broth feeds into a mechanical vapor recompression (MVR) evaporatorto remove water from the broth. Mechanical vapor recompressionevaporator and other types of evaporators that utilize vapor compressionto remove water from a liquid material and therefore concentrate theamount of 3HP (and salts thereof) that are contained in a liquid. Ifthermal heat such as steam is readily available, evaporators that usethermal heat (with or without vacuum) can also be used. An example of anevaporator that uses thermal heat from a source such as steam toconcentrate the 3HP equivalents in a broth are shell-in-tube evaporators(such as forced recirculation and falling film evaporators). Also,evaporators that utilize both mechanical vapor recompression and thermalheat cycle(s) from sources such as steam, or heated fluid (such as oil)may be effectively utilized. Two stages of MVR evaporation are used andoperate at 60-80° C. and 260-300 mm Hg. The concentrated broth exits theevaporator at 240 g/L 3HP equivalents.

It is also possible to concentrate the fermentation broth usingevaporation prior to the cell separation step. Concentrating the brothbefore the cell separation step reduces the total volume of brothfeeding into the centrifuge and does not require as many centrifugeunits given the smaller hydraulic loading of the evaporated broth. Cellremoval and evaporation are typically carried out before theacidulation. It is believed that the concentration of the 3HP and/orsalts thereof in the fermentation broth and absence of cells allows formore efficient acidulation and processing after acidulation, such asgypsum removal.

Acidulation:

Referring to FIG. 1, step 4, sulfuric acid is added to evaporatedconcentrated 3HP containing broth to lower the pH and convert most ofthe calcium salts of 3HP to the free acid form. The CaSO₄, formed (alsoknown as gypsum) precipitates out of solution. In a large agitated batchreactor, the sulfuric acid is added slowly to the concentrated 3HPcontaining broth, over the period of 15-25 minutes to slowly form gypsumprecipitate. The sulfuric acid is added until the concentrated brothreaches a pH of 1.7-1.8.

The acidulation step can be run as a batch or continuous process.Operating a continuous acidulation process has the advantage ofprocessing larger volumes of concentrated 3HP containing broth withoutthe need for an array of acidulation tanks. As an example of acontinuous acidulation step, for a broth flow of 115 m³/hr, theacidulation tank typically has a volume greater than 40 m³, typicallyrequiring a residence time of at least 20 minutes. The dosing rate ofsulfuric acid is controlled so the pH of the solution is between 1.6 and1.8.

Clarification to Remove Gypsum:

Referring to FIG. 1, step 5, the acidulated 3HP broth containing gypsumparticles is next clarified to remove gypsum. Combined with theacidulation step, the clarification step removes over 95% of the calciumin the broth prior to acidulation. The slurry of acidulated 3HP brothcontaining gypsum is fed onto a belt filter to filter out the gypsumparticles. A filter cake of gypsum forms on top of the filter as thefiltrate permeates the cake and filter. A vacuum is applied to the beltto help draw the broth through the filter. The gypsum cake does containresidual 3HP broth. Therefore, to increase the recovery of 3HP, water issprayed on the gypsum cake washing the broth away from the cake asfiltrate. To further increase recovery of 3HP, an additional stage ofwashing is performed and the filtrate is collected. To reduce the amountof wash water used, a counter current wash system is employed. Freshwater is applied to the 2^(nd) wash stage. The filtrate of that wash isused in the 1^(st) wash stage.

The gypsum cake thickness is typically maintained at 1 inch or thinnerso that the filtrate can readily permeate the cake and filter. As theconcentration of the 3HP in the evaporated fermentation broth feedingthe acidulation step increases, the permeation or flux rate of 3HPthrough the gypsum cake decreases. Table 10-1 shows the correlationbetween the 3HP equivalents concentration feeding acidulation and theresulting flux rate of broth through the filter. Typically, theevaporation step before the acidulation step is carried out in a mannerthat provides a broth concentrated in 3HP equivalents, wherein the 3HPequivalent concentration is below 350 g/L, preferably below 330 g/L.

TABLE 10-1 Flux rate of the broth through the filter cake whenacidification step is carried out on concentrated broths containingdiffering 3HP equivalents concentration. Cake thickness is close to 1inch thick. Flux is measured as ml of broth filtered per minute per cm²of surface area filter. 3HP equivalents (g/L) Flux (ml/min/cm²) 99 2.6158 2.4 199 1.7 231 0.8 279 0.2 356 <0.05

As can be seen from Table 10-1, the 3HP equivalent concentration of thebroth just prior to acidulation preferably is less than 330 grams 3HPequivalents/liter broth.

Other filtration devices such as a press filter or centrifuge could alsobe used to carry out the removal of gypsum (i.e. clarification step).

Demineralization/Ion Exchange:

Referring to FIG. 1, step 6, the clarified 3HP broth is next treated toreduce the total sulfate ions and phosphate ions present in the broth toproduce a reduced ion aqueous solution. The broth is passed through acation exchange column first to remove residual calcium as well as othercations such as potassium and manganese. As the capacity of the cationresin is consumed, some of the cations begin to breakthrough and exitwith the effluent 3HP containing solution. When this happens, water isfed through the column to elute out residual 3HP in the column. Thecolumn is then regenerated with HCl solution, rinsed with water, andthen put back into service.

The 3HP effluent out of the cation column is then fed into an anioncolumn where anions are removed, in particular phosphate and sulfateanions. Because the sulfate and phosphate concentrations can affect thesubsequent distillation steps, it is desirable to control theconcentration of sulfate and phosphate in the final output of the ionexchangers. Because of its charge density, phosphate ions tend tobreakthrough first. When sulfate levels start to break through, water isfed into the column to elute out anyresidual 3HP. The column isregenerated with a solution of NaOH, rinsed with water, and then putback into service.

The properties of the reduced ion aqueous solution comprising 3HP thatis obtainable from the above described demineralization described aboveare indicated in Table 10-2.

TABLE 10-2 Stream data for key components after each step of the 3HPrecovery process described above in example 9 and 10. Fermentation CellRemoval Evaporation Clarification Demineralization 3HP 80 g/L 75 g/L 240g/L 240 g/L 220 g/L equivalents concentration Ca 10000 ppm 10000 ppm25000 ppm 200-500 ppm 20 ppm Sulfate 200 ppm 200 ppm 400-700 ppm5000-10000 ppm 200 ppm Phosphate 300 ppm 300 ppm 500-800 ppm 500-800 ppm500 ppm pH 4.1 4.1 4.1 1.7 1.7

Table 10-2 shows that a 3HP containing reduced ion aqueous solution caneffectively be produced at large scale that has a total sulfate ion andphosphate ion concentration less than 3,000 ppm and in some instanceless than 1000 ppm.

Example 11

Now referring to FIG. 2, in this example, the 3HP containing reduced ionaqueous solution from Example 10 is fed to a three stage thermal vaporrecompression system. There, under the conditions of 0.39 bar pressureand each stage has a temperature of 80° C. to 85° C., the concentrationof the reduced ion aqueous solution is increased from 21 wt % 3HP (220g/L 3HP) to 62 wt % 3HP. Condensed water vapor from the evaporationprocess is recycled for use in fermentation and/or for flushing thedemineralization.

The concentrated 3HP containing solution is then fed to a series of twodistillation columns. In these columns, the 3HP and water exit in thedistillate, while impurities such as oligomers of 3HP and residualsugars exit from the bottoms of the columns. The first column operatesat a pressure of 35 mmHg and an internal temperature of 135° C. The feedto the second distillation column is the unvaporized bottom product fromthe first distillation step. The second column operates at a pressure of3 mmHg and a temperature of from 125° C. The distillates from thedistillation columns are condensed in a condenser against chilled water,resulting in a final product of 60% 3HP by weight.

This example (when compared with example 12 below) provides lowercapital and energy costs when compared to the example 12.

Example 12

Referring to FIG. 3, if higher overall recovery of 3HP from thefermentation broth is desirable at the expense of higher energy costsand higher capital costs, then example 12 is preferable. Example 12contains the same unit operations as Example 11, but order in which theoperations are carried out has been modified.

In this example, the 3HP containing reduced ion aqueous solution fromExample 10 is fed to a series of two distillation columns. In thesecolumns, the 3HP and water exit in the distillate, while impurities suchas oligomers of 3HP and residual sugars exit from the bottoms of thecolumns. The first column operates at a pressure of 35 mmHg and aninternal temperature of 135° C. The feed to the second distillationcolumn is the unvaporized bottom product from the first distillationstep. The second column operates at a pressure of 3 mmHg and atemperature of 125° C. to 150° C. The distillates from the distillationcolumns are condensed in a condenser against chilled water, resulting ina final product of 21% 3HP by weight. When compared to Example 11, theconcentration of 3HP is lower in the distillation column in Example 12.This allows the column to operate at lower temperatures and impacts thethermodynamics to increase the overall recovery of 3HP. The lowerconcentration also means that additional water must be vaporized intothe distillate (leading to increased energy costs) and a larger totalunit is required to vaporize a larger volume.

The distilled broth is then fed to a three stage thermal vaporrecompression system. There, under the conditions of 0.39 bar pressureand each stage has a temperature of 80° C. to 85° C., the concentrationof the demineralized broth is increased from 21 wt % 3HP to 60 wt % 3HP.Condensed water vapor from the evaporation process is recycled for usein fermentation and/or for flushing the demineralization.

Example 13

The distillation step of either Example 11 or Example 12 may be modifiedby adding an optional steam stripping. In steam stripping, steam isadded to the bottom of the distillation column, providing additionalmass into which 3HP can vaporize. This has the effect of increasing theoverall recovery of 3HP from the process, while increasing energy usage,and reducing the final concentration of the recovered 3HP. Steamstripping would typically be utilized with a 3HP containing reduced ionaqueous solutions having at least 40 wt % 3HP, preferably at least 50 wt% 3HP.

As an example, but in no way limiting its use, FIG. 4 demonstrates theeffect of adding stripping steam to total recovery on a distillationcolumn with a 60 wt % 3HP feed. FIG. 4 shows that as the feed rate ofsteam introduced into a distillation system is increased (relative tothe feed rate of 3HP containing solution), the percent of overall 3HPthat is recovered in the distillate increases. However, in general asratio of steam feed rate:3HP feed rate increases, the energy usagerequired increases and the concentration of 3HP in the distillatedecreases.

The energy costs and yields obtained from Examples 11 and 12, with andwithout steam stripping, are listed in Table 13-1 below:

TABLE 13-1 Comparison of 3HP recovery and energy usage based on theconfiguration of the evaporation and distillation steps. Change in 3HPEnergy Usage in Recovery evaporation and Final 3HP (% Relativedistillation (Relative to concentration Example to Example 11) Example11) (wt %) Example 11 0% 1.0 60 wt % Example 12 3% 1.75 60 wt % Example11 3% 1.2 55 wt % with Steam Stripping

Table 13-1 shows that the relative recovery of 3HP from a method thatuses evaporation before distillation is slightly lower than the recoveryfrom a method first distills the 3HP and then increases theconcentration using evaporation. However, as can be seen from Table13-1, a system that uses a distillation step to separate 3HP from othercomponents of a 3HP containing reduced ion aqueous solution and thenutilizes evaporation to increase the concentration of the 3HP, utilizesfar more energy than a comparable system as described in Example 11.

All patents, patent applications (including provisional applications),and publications cited herein are incorporated by reference as ifindividually incorporated for all purposes. Unless otherwise indicated,all parts and percentages are by weight and all molecular weights areweight average molecular weights. The foregoing detailed description hasbeen given for clarity of understanding only. No unnecessary limitationsare to be understood therefrom. The invention is not limited to theexact details shown and described, for variations obvious to one skilledin the art will be included within the invention defined by the claims.

1. A method for recovering a composition enriched in 3-hydroxypropionicacid from a fermentation broth comprising 3-hydroxypropionic acid and/orsalts thereof, the method comprising the steps of: (a) providing thefermentation broth having a pH of from about 2 to about 8 comprising:3-hydroxypropionic acid and/or salts thereof, and a total sulfate ionand phosphate ion concentration; (b) acidifying the fermentation brothto lower the pH to from about 1 to about 3 to form an aqueous solutioncomprising 3-hydroxypropionic acid; (c) reducing the total sulfate ionand phosphate ion concentration of the aqueous solution to produce areduced ion aqueous solution comprising 3-hydroxypropionic acid; (d)distilling the reduced ion aqueous solution at a pH of from about 1 toabout 3 by applying vacuum and heat to the reduced ion aqueous solutionto form an aqueous distillation product comprising 3-hydroxypropionicacid; and (e) recovering the aqueous distillation product comprising3-hydroxypropionic acid at a concentration of at least twenty percent byweight of the aqueous distillation product and wherein the aqueousdistillation product comprises less than five parts by weight acrylicacid per one hundred parts by weight 3-hydroxypropionic acid present.2.-19. (canceled)
 20. The method of claim 1, wherein the step (d) ofdistilling the reduced ion aqueous solution is carried by equipmentselected from wiped film evaporation equipment, rising film evaporatorequipment, thin film evaporation equipment, (centrifugal) moleculardistillation equipment, and falling film distillation equipment. 21.-34.(canceled)
 35. The method of claim 1, further comprising (f) recoveringa distillation bottom stream from distilling step (d), the distillationbottom stream comprising from about 5 percent to about 20 percent3-hydroxypropionic acid of the total amount of the 3-hydroxypropionicacid present in the reduced ion aqueous solution of step (c); (g)applying vacuum and heat to the distillation bottom stream; and (h)recovering a second aqueous distillation product comprising3-hydroxypropionic acid, wherein the second aqueous distillation productcomprises less than about 3 part by weight acrylic acid per one hundredpart by weight 3-hydroxypropionic acid present in the second aqueousdistillation product.
 36. The method of claim 35, wherein water is addedto the distillation bottom stream prior to step (g). 37.-48. (canceled)